This invention concerns methods of production and compositions for non-covalently complexed multivalent proteosome vaccines for mucosal and parenteral administration.
In order for multivalent sub-unit vaccines to stimulate optimal immune responses to each of the components, the proper components should be appropriately associated and each be available to the immune system so that they may be efficiently recognized and processed by cells of the immune system. Prime examples of such non-covalently complexed vaccines include proteosome vaccines which can consist of neisserial outer membrane protein proteosomes non-covalently complexed to a wide variety of antigens including peptides, lipopeptides, transmembrane or toxoided proteins, polysaccharides or lipopolysaccharides (LPS) (patent application Ser. No. 07/065,440 filed Jun. 23, 1987 xe2x80x9cImmunogenic peptide vaccines and methods of preparationxe2x80x9d; Ser. No. 07/336,952 filed Apr. 12, 1989 Immunopotentiaing system for large proteins and polypeptidesxe2x80x9d; Ser. No. 07-958,426 filed Oct. 8, 1992 xe2x80x9cOral or Intranasal Vaccines Using Hydrophobic Complexes Having Proteosomes and Lipopolysaccharidesxe2x80x9d; Ser. No. 08/029,666 filed Mar. 11, 1993 xe2x80x9cImmunopotentiating Systems for Preparation of Immunogenic Materialsxe2x80x9d; Ser. No. 08/143,365 filed Oct. 29, 1993 xe2x80x9cImmunopotentiating Systems for Preparation of Immunogenic Materialsxe2x80x9d; Ser. No. 93/10,402 filed Oct. 29, 1993 xe2x80x9cSubmicron Emulsions as Vaccine Adjuvantsxe2x80x9d; Ser. No. 08/063,613 filed May 18, 1994 Solid Fat Nanoemulsions as Vehicles for Vaccine Deliveryxe2x80x9d and publications Orr, N., Robin, G., Cohen, D., Arnon, R. and Lowell, G. H. (1993). Immunogenicity and Efficacy of Oral or Intranasal Shigella flexneri 2a and Shigella sonnei Proteosome-Lipopolysaccharide Vaccines in Animal Models. Infect. Immun. 61:2390; Mallett, C. P., T. L. Hale, R. Kaminski, T. Larsen, N. Orr, D. Cohen, and G. H. Lowell. 1995. Intranasal or intragastric immunization with proteosome-Shigella lipopolysaccharide vaccines protect against lethal pneumonia in a murine model of shigellosis. Infect. Immun. 63:2382-2386.; Lowell G H, Kaminski R W, Grate S et al. (1996) Intranasal and intramuscular proteosome-staphylococcal enterotoxin B (SEB) toxoid vaccines: immunogenicity and efficacy against lethal SEB intoxication in mice. Infec. Immun. 64:1706-1713.; Lowell, G. H. (1990) Proteosomes, Hydrophobic Anchors, Iscoms and Liposomes for Improved Presentation of Peptide and Protein Vaccines. in New Generation Vaccines: G. C. Woodrow and M. M. Levine, eds. (Marcel Dekker, NY). Chapter 12 (pp. 141-160) and Lowell, G. H., W. R. Ballou, L. F. Smith, R. A. Wirtz, W. D. Zollinger and W. T. Hockmeyer. 1988. Proteosome-lipopeptide vaccines: enhancement of immunogenicity for malaria CS peptides. Science 240:800.)
The contents of all the documents cited herein are expressly incorporated by reference.
For practical application in administering vaccines to protect against disease, it is frequently necessary to deliver several such antigens at the same time usually due to the fact that individuals are susceptible to the contraction of diseases caused by a variety of organisms. Moreover, several organisms, whether or not they are related to one another, often are endemic in the same location and therefore individuals requiring protection may need vaccination with several types of vaccines.
In the past, the production of vaccines that require non-covalent complexing of components has been accomplished using simple dialysis in which components are placed in dialysis tubing in the presence of dialyzable detergent and the mixture is dialyzed for 7-10 days to attempt to remove the detergent. The practical disadvantages of this system tend to severely preclude the advanced development and commercialization of this technology for several reasons including 1) Time: Length of time of the procedure: The need to use GMP resources for weeks while the vaccine is dialyzing is impractical both due to the excess costs involved and the increased opportunity for breakdown or contamination of mechanical or biological components during this extended period of time; 2) Contamination: Increased opportunity for contamination: dialysis tubing is difficult to sterilize, dialysis tubing requires manually opening and closing the system thereby exposing the components to contamination during both the loading and unloading process. Since many days transpire between loading and unloading the tubing, the risk of a small contamination in the initial days of the process may readily be magnified during the many days of dialysis to render this method useless for practical vaccine manufacture. The risk of puncturing the bag can result in loss of product. 3) Temperature: Necessity to perform the dialysis at 40xc2x0 C. due to the extensive time involved; 4) Volume of dialyzing fluids: In order to manufacture vaccine for scale-up of the process, the use of massive amounts of dialysis fluid would be necessary since a 200:1 ratio of liquid outside to the dialysis tubing to inside the tubing is typically required. Therefore, for example, the production of a pilot lot of two liters of vaccine would require 400 liters of fluid outside the tubing per dayxe2x80x944,000 liters per 10 daysxe2x80x94and the production of a production lot of 20-200 liters would require 40,000-4,000,000 liters. These amounts are wasteful and impractical compared to the method used in the instant invention; Dialysis tubing is not scalable since large amounts of product is problematic and 5) Inability to readily measure completeness of removal of the detergent so as to maximize vaccine effectiveness. Since the dialysis bag is placed in a container with 200 volumes of buffer, the ongoing measurement of detergent removal is neither practical nor feasible and 6) In addition, no method has been described for the measurement of the presence of the detergent used in the preferred embodiment, Empigen BB.
The second problem solved in this invention is the demonstration of the method of producing and delivering multivalent vaccines. Components can either be made together or produced separately and mixed together prior to administration. The instant invention demonstrates the optimal way of preparing such multivalent vaccines.
The subject of the instant invention broadly relates to the production and manufacture of proteosome-amphiphilic determinant vaccines designed for either parenteral or especially for mucosal administration including , but not limited to, respiratory (e.g. including intranasal, intrapharyngeaeal and intrapulmonary), gastro-intestinal (e.g. including oral or rectal) or topical (e.g. conjunctival or otic) administration to induce both systemic (serum) and mucosal (including respiratory and intestinal) antibody responses. An amphiphilic determinant is a molecule having hydrophobic and hydrophilic regions which, when appropriately formulated with proteosomes, align with the proteosomes to for a complex which elicits an immunologic response in a subject. Typical amphiphilic determinants include glycolipids, liposaccharides (including detoxified lipopolysaccharides), lipopeptides, transmembrane, envelope or toxoided proteins, or proteins or peptides with intrinsic hydrophobic amino acid anchors. These determinant materials can be obtained from gram negative bacteria including eschefichia, klebsiella, pseudomonas, hemophilus brucella, shigella and neisseria. More specifically, the invention relates to proteosome vaccines in which meningococcal outer membrane protein proteosome preparations (prepared from any strain of N. meningiditis or N. gonorrhea or other neisserial species) are non-covalently complexed to native or detoxified shigella or neisserial lipopolysaccharides or lipooligosaccharides to form vaccines designed to protect against diseases caused by gram negative organisms that contain any of the component parts of the complex including meningococci or shigellae. More specifically, the invention relates to proteosome vaccines that contain LPS that induce antibody responses that recognize type-specific somatic polysaccharide O-antigens of shigella lipopolysaccharides and thereby confer homologous protection against shigellosis. Still more specifically, the lipopolysaccharides that, when complexed to proteosomes induce such anti-shigella protective immune responses are prepared and purified from either Shigella sonnei or Plesiomonas shigelloides for immunity against Shigella sonnei disease, from Shigella flexneri 2a for immunity to Shigella flexneri 2a disease, and so forth, using LPS derived from homologous or antigenically cross-reacting organisms to confer homologous immunity against shigellosis caused by S. flexneri 2a (or 3a etc.), S. boydii, S. sonnei etc. Still more specifically, the instant invention describes the successful administration of proteosome-shigella vaccines that are multivalent in that two independently made proteosome vaccines using shigella LPSs derived from S. flexneri 2a (for S. flexneri 2a disease) and from P. shigelloides or S. sonnei (for S. sonnei disease) are administered together thereby inducing antibodies that recognize the two organisms and thereby conferring protection against the two types of diseases. Most specifically, the instant invention relates to a proteosome-shigella LPS vaccine in which proteosomes from group B type 2b meningococci are complexed to P. shigelloides LPS using hollow fiber diafiltration technology to produce a vaccine administered by mucosal respiratory and/or gastro-intestinal routes to induce antibodies that recognize the somatic O-antigen LPS of S. sonnei and thereby protect against shigellosis caused by this organism. Other conventional ultrafiltration/diafiltration are envisioned, e.g. platform membrane and membrane cartridge.
The present invention provides methodology to produce non-covalently complexed vaccines in a manner that 1) Decreases the time required, 2) Decreases the opportunity for contamination, 3) Increases the temperature to ambient temperature that such vaccines can be produced, 4) Allows for efficient scale-up of the production process so as to require minimum use of reagents, 5) Allows for reliable and efficient sampling of dialysate so as to be able to repeatedly measure rate of removal of the detergent so as to optimize efficiency of the operation. This leads directly to an increase in the complexing efficiency of vaccine so as to produce vaccine with measurably greater immunogenicity at lower doses. In this manner, the overall quality of the product is significantly enhanced. 6) In addition, a method is described to measure the presence of the detergent used in the preferred embodiment, Empigen BB. Other dialyzable detergents can be used in place of Empigen BB. Furthermore, using the method of the instant invention, it has been demonstrated that the vaccine can be lyophilized and re-hydrated in such a manner as to retain optimal vaccine potency as measured by vaccine immunogenicity.
The method of the instant invention entails the use of a hollow fiber ultrafiltration/diafiltration cartridge to effect complexing by removal of the detergent. By varying the size of the housing of the cartridge, the time of dialysis for production of a lot of vaccine can be reduced from  greater than 7-10 days to less than 72 hours and usually less than 48 or 24 hours. Since this short time is used, the reaction temperature can be increased from 4xc2x0 to normal room temperature without compromising the process or the integrity of the products. While the process was performed at about 20xc2x0 C., temperatures between 0xc2x0 and 40xc2x0 C. are contemplated. Since the system is closed, there is a highly reduced potential for contamination. In addition, by increasing the size of the housing, an exceedingly large amount of material can be processed in a relatively short period of time thereby allowing for efficient and reproducible scale-up of the procedure for commercial development. Furthermore, the permeate can be repeatedly sampled to measure the removal of the detergent as can be accomplished using the test of the instant invention to measure the presence of the detergent empigen BB, the detergent used in the preferred embodiment. The uniqueness of this methodology needed experimental verification since it was not obvious that the complexing and the structure of the vaccine into immunogenic materials that was accomplished by slowly dialyzing over 7-10 days using a stationary dialysis bag could be equalled or improved upon using the hollow fiber technology in which the moieties to be complexed are moving through tubes at exceedingly high flow rates compared to that of the stationary dialysis tube.
Since the nominal molecular weight cutoff of the membrane used can be selected to be from 1,000, 3,000, 5,000, 10,000, 30,000, 50,000 or greater, depending on the size of the components to be non-covalently complexed, this system is readily adaptable to the complexing of native or detoxified lipopolysaccharides, lipids, peptides, lipopeptides, liposaccharides, polysaccharides, gangliosides or transmembrane, envelope or native or toxoided proteins to each other or to meningococcal outer membrane protein preparations of proteosomes.
The resultant product can be used as a vaccine administered either parenterally or mucosally i.e. via the respiratory or gastro-intestinal tract e.g. intranasally, orally, by oropharyngeal inhalant, topically or rectally. In the example given, it is shown that proteosomes are non-covalently complexed to P. shigelloides LPS to form a vaccine that induces the anti-S. sonnei LPS responses necessary for protection against S. sonnei shigellosis.
The methodology has three stages: a preliminary operation; complexing the proteosome with an amphiphilic determinant e.g. P. Shigelloides LPS; followed by a sterile filtration.
To optimally deliver multivalent vaccines, the present invention shows that the best method is to make the specific vaccines individually and then immunize with the two individually made vaccines, each at the optimal concentration, on the same day. Other possibilities such as making hybrid vaccines during the formation of the non-covalent complexes have also been accomplished or are envisioned.